Green biosynthesis of gold nanometre scale plates using the leaf extracts from an indigenous Australian plant Eucalyptus macrocarpa
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In this preliminary study, we demonstrate an environmentally friendly process for the green synthesis of gold nanometre scale particles using the leaf extract from an indigenous Australian plant Eucalyptus macrocarpa as both the stabilising agent and the reducing agent. The synthesis process is straightforward, clean and non-toxic. It also has the advantages of being performed at room temperature and does not need complex processing equipment. Formation of the gold nanometre sized particles was confirmed and characterised by UV-visible spectroscopy, X-ray diffraction, transmission electron microscopy and field emission scanning electron microscopy. The antibacterial activity of the synthesised gold particles was also quantified using the sensitivity method of Kirby–Bauer.
KeywordsGold Nano-particles Green synthesis Antibacterial Eucalyptus macrocarpa
Metallic nanometre scale particles, in particular colloidal gold nanoparticles (Au NPs), have attracted considerable interest in many fields such as medicine, biotechnology, materials science, photonics and electronics [1–4]. The size, shape and surface morphology can have a profound influence on the chemical, physical, optical and electronic properties of nano-materials [5, 6]. This is indeed the case for a noble metal such as Au, where the metal exhibits a strong surface plasmon resonance when exposed to electromagnetic radiation . Until recently, producing nanoparticles involved expensive chemical and physical processes that often used toxic materials with potential hazards such as environmental toxicity, cytotoxicity and carcinogenicity . An attractive alternative to the traditional manufacturing techniques used for the production of nanoparticles involves using a green environmentally friendly technology based on biological systems such as plants [9, 10], bacteria [11, 12], fungus [13, 14] and similar organisms [15, 16]. Synthesising nanoparticles via biological systems offers a clean, nontoxic and environmentally friendly method with the potential to deliver a wide variety of nanoparticle types, sizes, shapes and morphologies. Out of the several biological systems mentioned above, synthesising nanoparticles via leaf extracts from plants is a relatively straight forward technique. The technique does not need any special culture preparation or isolation techniques that are normally required for bacteria and fungi based synthesis techniques.
Au NPs have attracted significant interest over the last decade as a potential platform for a number of biomedical applications such as biosensors , clinical chemistry , fluorescent labelling for immunoassays , tumour destruction via heating (hyperthermia) , targeted delivery of therapeutic drugs and genetic substances  and as antibacterial drugs [22, 23]. The cells of most living organisms have dimensions in the range of 10 to 50 μm, while the cells' internal organelles and other sub-cellular structures are all in the sub-micron size range. The difference in size between a typical cell and a nanometre scale particle can range from a hundred times up to ten thousand times. This significant difference in size range between the cell and a nanometre scale particle gives the NP the potential to biophysically interact with biological molecules both inside and on the surface of cell . Recent studies have shown that membrane damage and toxicity can result from the biosorption and cellular uptake of NPs by bacteria [24, 25]. However, the processes behind NP inhibition of bacterial growth are not fully understood, but some studies have strongly suggested that the size, shape and surface modifications could influence the antibacterial properties of the NPs [25, 26].
Chrysotherapy is a branch of medicine that uses Au as a medicinal compound in the treatment of certain diseases such as rheumatoid arthritis. In addition, Au NPs have also been successfully used as a carrier platform for the delivery of double-stranded DNA in gene gun technology . Another feature of Au NPs is their ability to passively accumulate in tumours, where their good optical and chemical properties can be used in thermal treatment therapies [28, 29]. Moreover, recent studies have shown that cancer drugs bonded to the surface of Au NPs can be more effectively targeted to tumours, thus improving delivery and minimising treatment durations and side effects of anticancer drugs [3, 30–33]. Au NPs have also attracted significant scientific interest as a new class of biomedical materials, since they can act as both antibacterial and antifungal agents capable of interacting with microorganisms to produce cellular damage . The ability to induce cellular damage and the ultimate death of various strains of bacteria and yeast microorganisms has many significant health benefits. For example, both bacterial and fungal species have the ability to develop immunity against commonly used antibiotic with time. Therefore, new and more effective antimicrobial agents are needed to fight antibiotic resistant strains of microorganisms. Au NPs have the potential to be an effective antimicrobial agent, especially if they can be synthesised via a green chemistry route that ensures a clean, nontoxic and environmentally friendly method for producing NPs.
In this paper, we report for the first time the green synthesis of stable Au NPs by the direct reduction of AuCl4 − ions via Mottlecah leaf extracts without using conventional stabilising ligands. The advantages of using this approach include: (1) the leaf extract acts as both stabilising agent and reducing agent during the synthesis process; (2) the aqueous synthesis process is environmental friendly and produces no toxic waste; and (3) the technique is simple, straight forward and does not require specialised equipment. Furthermore, the antibacterial activity of both the synthesised Au NPs and the leaf extracts were tested against Bacillus subtilis and Escherichia coli using the Kirby–Bauer sensitivity method .
Materials and methods
The AuCl4 − ions were produced by dissolving high purity Au wire (99.99 %) in a solution of aqua regia. Both HNO3 and HCl were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia) and used without further purification. Milli-Q® water was used throughout all synthesis procedures involving aqueous solutions and was produced by a Barnstead Ultrapure Water System D11931 (Thermo Scientific Dubuque IA 18.3 MΩ cm−1).
Leaf material and preparation of leaf extract
The E. macrocarpa leaves were collected from several locations around the Murdoch University campus in Perth, Western Australia. A wide selection of Mottlecah leaves, ranging from young to mature leaves, was harvested from various locations on each plant. Generally, five locations were selected (top, north, south, east and west), and on average, ten leaves were taken from each of the locations. Both the adaxial and abaxial sides of the leaf were examined, with only healthy leaves free from damage being harvested. The leaves were washed several times with Milli-Q® water to remove any dust or debris. After cleaning, 10 g of Mottlecah leaves were finely cut into small strips and added to a 100 mL solution of Milli-Q® water. The aqueous mixture was then poured into the blending bowl of an IKA® T25 Digital Ultra Turrax® homogenizer. The mixture was homogenised at 5,000 rpm for 10 min at a room temperature of 24 °C. At the end of this time, the solution was filtered using a Hirsch funnel to remove leaf debris. This was followed by two further filtrations using a 0.22 μm Millex® (33 mm diameter) syringe filter unit. At the end of the three-step filtration procedure, the leaf extract was placed in a glass vial ready for the synthesis of Au NPs.
Removal of epicuticular waxes
To determine the influence of epicuticular wax in the leaf extract, it was necessary to prepare a leaf extract without wax. This was achieved by first selecting several pre-cleaned leaves (discussed above) and then using a solution of chloroform to wash each leaf. Both the adaxial and abaxial sides of the leaf were washed to remove all surface waxes. The washing procedure consisted of slowly pouring a 50 mL solution of chloroform over the entire inclined leaf collecting the run off in a small beaker, each side being done in turn. During the 30 s washing period, the chloroform efficiently removed the epicuticular waxes from the leaf surface. Subsequent scanning electron microscope (SEM) analysis revealed that the waxes had indeed been removed from the leaf surface leaving the underlining cutin undamaged.
Synthesis of gold nanoparticles
The Au NPs used as the control were synthesised by first adding a 1.0 mL solution of 1 mM AuCl4 − to a 10 mL solution of Milli-Q® water, while the solution was stirred vigorously. This was followed by adding a 1.0 mL solution of 1 mM sodium citrate (stabilising and capping agent) to the aqueous solution at room temperature (24 °C). The reduction of the Au nanoparticles was initiated by the addition of a 1.0 mL solution of 0.01 M sodium borohydride to the aqueous solution, while the solution was stirred. The reduction process was allowed to proceed at room temperature (24 °C).
The biological reduction of a 1.0 mL solution of 10 mM aqueous AuCl4 − ion solution was investigated using three solutions with varying amounts of leaf extract without wax and with wax. The quantities of leaf extract used to make up the solutions consisted of 1 mL for g1, 2 mL for g2 and 3 mL for g3. Once the AuCl4 − ion solution was added to each quantity of leaf extract, the solutions were then vigorously stirred for 1 min. The reduction process was allowed to proceed at room temperature (24 °C), with stable Au NPs being produced within 1 h.
A clean Mottlecah leaf was selected. The leaf was laid flat in the horizontal plane to prevent the various droplet types from rolling off the surface. Then using a fluid-specific clean glass pipette fitted with a rubber bulb, four individual droplets were placed onto the leaf surface. The droplets consisting of: (1) filtered solution containing Au NPs, (2) solution s1 composed of AuCl4 − and leaf extract solution, (3) pure AuCl4 − solution, and (4) solution of raw leaf extract. The progress of the reduction process was photographed over a 2-h period using a Canon EDS 600 D digital camera (Canon Inc, Tokyo Japan fitted with macro lens EF 100 mm 1:2:8 USM). A typical photograph taken after 1 h is presented in Fig. 1b.
Characterisation of biologically reduced Au nanoparticles
All samples were examined and characterised using five advanced analysis techniques. These included: UV-visible spectrum analysis, X-ray diffraction spectroscopy (XRD), energy dispersive X-ray spectroscopy (EDAX), transmission electron microscopy (TEM) and field emission scanning electron microscopy (FESEM).
UV-visible spectrum analysis
A series of samples were prepared. The first set consisted of three controls: (1) Milli-Q® water, (2) pure AuCl4 − solution and (3) pre-filtered pure leaf extract (filtered twice, each time using a new Whatman 0.22 μm syringe filter). The test solutions consisted of the three Au colloids g1, g2 and g3. The UV-visible spectra of each of the samples was then measured using a Varian Cary 50 series UV-visible spectrophotometer version 3, over a spectral range from 200 to 1,100 nm, with a 1 nm resolution over the first hour at room temperature of 24 °C.
After the end of each reduction procedure, samples for XRD examination were extracted from each glass vial using a clean glass pipette fitted with a rubber bulb. Then two to three drops of each sample were dispersed over the surface of a specific glass microscope slide. Then each glass slide was then dried under vacuum for a period of 4 h. At the end of this time, the dried samples were then characterised using XRD spectroscopy. The XRD spectra were recorded at room temperature (22 °C), using a Bruker D8 series diffractometer (Cu Kα = 1.5406 Å radiation source) operating at 40 kV and 30 mA. The diffraction patterns were collected over a 2θ range from 15 to 80°, with an incremental step size of 0.04° using flat plane geometry. The acquisition time was 2 s. The powder XRD spectrum was used to identify the size of the Au particles and their crystalline structure. The particle size was calculated using the Debye–Scherrer equation (Eq. 2) from the respective XRD patterns and estimated from both TEM and FESEM images.
Each sample for EDAX examination was initially deposited onto a thin mica strip using a glass pipette, the mica strip was attached to a SEM stub using carbon tape. The samples were then dried under vacuum overnight. The following day, all samples were sputter-coated with a 3-nm layer of platinum. The samples were then examined using an Oxford Instruments EDS X-ray detector (EDAX) and Oxford Instruments energy dispersive X-ray detectors (EDS). The electron backscatter diffraction was used during the analysis, and the EDS aperture was set to 60 μm and operated at 20 kV.
The size and morphology of the Au NPs was investigated using TEM. Sample preparation consisted of filtering the suspensions two times, each time using a new Whatman 0.22 μm syringe filter. After filtration, a single drop from each sample was deposited onto its respective carbon-coated copper TEM grid using a micropipette and then allowed to slowly dry over a 24-h period. After sample preparation, a bright field TEM study was carried out using a Phillips CM-100 electron microscope (Phillips Corporation Eindhoven, The Netherlands) operating at 80 kV.
Each sample for FESEM examination was initially deposited onto a thin mica strip using a glass pipette; the mica strip was attached to a SEM stub using carbon tape. The samples were then dried under vacuum overnight. The following day, all samples were sputter-coated with a 3-nm layer of platinum. The particle size and morphological features of the samples were investigated using a high resolution FESEM (Zeiss Neon 40EsB FIBSEM) at 5 kV, with a 30-μm aperture operating under a pressure of 1×10−10 Torr.
Microbial sensitivity of Au nanoparticles
The antibacterial activity of the biologically reduced Au NPs was investigated using the sensitivity method of Kirby–Bauer . In this technique, the test media was composed of Difco nutrient agar (23 g/1,000 mL), agar (5 g/1,000 mL), yeast extract (4 g/1,000 mL) and distilled water (1,000 mL). The components were thoroughly mixed via heating and agitation to completely dissolve the components. The medium was then autoclaved at 121 °C for 15 min. At the end of the autoclaving period, a 20-mL volume of the media was added to each Petri dish (90 mm diameter) and then allowed to solidify at room temperature before being stored at 4 °C for future use.
Two bacteria, namely E. coli and B. subtilis were used to investigate the antibacterial activity of the Au NPs. In each case, the respective bacteria were cultured in a media composed of Difco nutrient broth (8 g/1,000 mL), yeast extract (4 g/1,000 mL) and distilled water (1,000 mL). The nutrient medium was then autoclaved at 121 °C for 15 min. After autoclaving, the nutrient medium was ready for culturing bacteria; in the first stage, each respective bacteria was added to a 10-mL sample of the nutrient medium and then incubated overnight at 28 °C while being agitated. In the second stage, the cultures were then added to a 100-mL solution of nutrient media and further incubated overnight at 28 °C, while being agitated. The following day, a 1-mL sample of the culture medium was pipette onto the surface of the nutrient containing Petri dish. The flooded dish is then carefully tilted in various orientations, until the entire nutrient surface was covered. Once the nutrient surface was covered, the excess culture medium was carefully removed using a pipette. Then, the cover was placed on each Petri dish, inverted and left to dry for 45 min.
While the Petri dishes were drying, 25 μg/mL solutions of as prepared Au NPs (Au NPs control and leaf extract solution (with and without wax) and leaf extract + Au NPs solution g1 (with and without wax)) were transferred onto a sterile 6-mm diameter Whatman AA (2017–006) sample disc using a pipette, and then allowed to dry for 20 min. After drying, sterile forceps were used to place the sample disc onto the centre of the inoculated Petri dish. After all the samples were placed in their respective inoculated dishes, the covers were fitted and then the dishes were incubated overnight at 28 °C. After incubation, the diameter of the bacterial inhibition zone of each sample was measured using a standard scale ruler with an accuracy of ± 0.5 mm, with all measurements being rounded up to the next whole millimetre. All sample experiments were carried out in triplicate and the mean inhibition zone diameters recorded.
In addition, optical microscopy was used throughout the studies to examine the bacterial inhibition zone. An Olympus BX51 compound microscope (Olympus Optical Co. Ltd., Tokyo, Japan) was used for all optical studies, and photographs were taken using the DP 70 camera attachment.
Results and discussions
A possible mechanism to explain the plate-like growth and morphology of particles during reduction in the leaf extract could be the result of competitive growth between crystallographic surfaces, which results in a preferential attachment in a particular orientation . An alternative mechanism proposed by Wang et al. arises from the initial spontaneous self-assembly of particles along a particular crystallographic orientation. This is followed by other Au particles coalescing at the planar interface, which effectively reduces their surface energy and contributes to the growth of the particle along this particular orientation . In this study, analysis of the XRD data clearly indicates the dominance of the (111) orientation and confirms preferential particle growth along (111) crystal plane. This explains the inherent anisotropy of the atomically flat surface of the Au crystalline-shaped plates produced initially in the nanometre range. Subsequent growth in the micrometre range also produces the atomically flat surfaces, which are characteristic of the plate-like particles synthesised using E. macrocarpa leaf extracts.
In the case of the Au NPs, the first tested was the Au NP (control), which was synthesised using sodium borohydride as the reducing agent and sodium citrate as the stabilising and capping agent. The Petri dishes containing a sterile pad treated with Au NPs (control) recorded a zone of inhibition of 12 mm for both B. subtilis and E. coli. The results of the zone of inhibition measurements indicate that both bacteria are resistant to the Au NP control. In the next step, Au NPs synthesised using the leaf extract were tested against the two bacteria. The Petri dishes containing a sterile pad treated with Au NPs+leaf extract (without wax) recorded a zone of inhibition of 16 mm for B. subtilis and 19 mm for E. coli. Since the leaf extract without wax was found to have no zone of inhibition (no antimicrobial effect), the larger zones of inhibition recorded for both bacteria are the result of the bio-reduced Au particles. This result indicates that both bacteria are more sensitive to the bio-reduced Au particle. And in the final test set, Petri dishes containing a sterile pad treated with Au NPs+leaf extract (with wax) recorded a zone of inhibition of 20 mm for B. subtilis and 29 mm for E. coli. The results of the Au NPs (control) and Au NPs synthesised using leaf extract are graphically presented in Fig. 5c (B. subtilis) and d (E. coli).
Inspection of both Fig. 5c and d reveals that the size of the diameters obtained for the zone of inhibition are much larger for Au NPs+leaf extract (with wax) than those without leaf extract. It is also evident that there is a synergistic effect between the Au NPs and the leaf extract. This is dramatically seen for Au NPs+leaf extract (with wax) against E. coli (zone of inhibition is 29 mm), which was significantly greater than the 19 mm for Au NPs+leaf extract (without wax). The results also show that Au NPs+leaf extract (with wax) was much more effective against the gram-negative E. coli (diameter 29 mm) than the gram-positive B. subtilis (diameter 20 mm). A photograph of the 29-mm zone of inhibition for E. coli resulting from Au NPs+leaf extract (with wax) is presented in Fig. 5a, while an enlarged microscopic view of the interface between E. coli and the zone of inhibition is presented in Fig. 5b. The results of the antibacterial study reveal that both B. subtilis (gram-positive) and E. coli (gram-negative) were sensitive to Au NPs+leaf extract (with wax). The leaf extract without wax proved to have no antibacterial effect. However, leaf extract with wax produced a small zone of inhibition (9 mm) for both bacteria, indicating they were both resistant to the leaf extract. The results indicate that the antibacterial properties contained within the leaf are resident in the waxes, and when the waxes are combined with Au NPs, a significant synergistic effect was produced. Combining the inherent properties of the leaf wax with those of Au NPs has proven to be beneficial in producing an effective antibacterial agent against both B. subtilis and E. coli.
A straightforward, clean and environmentally friendly method of biologically synthesising Au NPs was demonstrated using the leaf extracts from an indigenous Australian plant E. macrocarpa. The leaf extracts acted as both the stabilising agent and the reducing agent, with the process occurring at room temperature. The biological reduction of AuCl4 − produced an aggregate of NPs consisting of spherical particles ranging in size from 20 to 80 nm and nanometre-sized crystalline shapes (equilateral or truncated triangular, pentagon and hexagonal), ranging in size from 50 to 100 nm. Longer reduction times allowed the NPs to grow into micrometre-sized particles of varying size and morphology. The sensitivity method of Kirby–Bauer was used to establish a synergistic effect between the Au NPs and the waxes contained in the leaf extracts. And when combined, the Au NPs and leaf extracts (with wax) created an effective antibacterial agent against both B. subtilis and E. coli.
This work was partly supported by the Western Australian Nanochemistry Research Institute (WANRI). The authors would also like to thank Mr Ravi Krishna Brundavanam for his assistance with the XRD measurements and Mrs. Monaliben Shah, Mrs. Sridevi Brundavanam and Dr Michelle Buttery for their assistance in the antibacterial studies.
Conflict of interest
The authors claim no conflict of interest in this work.
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